Carotenoid pigments can be grouped into one or the other of two C40 families: the hydrocarbon carotenes or the oxygenated xanthophylls. The xanthophylls that are derivatives of β-carotene typically contain a hydroxyl or keto group or both in one or both of the β-ionene rings present in the xanthophyll molecule, thereby providing a number of possible structurally similar compounds. Xanthophylls containing only hydroxyl groups tend to be yellow pigments, whereas those containing at least one keto group tend to be redder pigments.
Astaxanthin [3,3′-dihydroxy-β,β-carotene-4,4′-dione; structural formula below] is a xanthophyll carotenoid that occurs particularly in a wide variety of marine animals including red fishes such as sea bream and salmon, and crustaceans such as crab, lobster, and shrimp.

Because animals generally cannot biosynthesize carotenoids, they obtain those carotenoids present in microorganisms or plants upon which they feed. For this reason, astaxanthin has been widely used as a feed additive in aquaculture for the purpose of red color enhancement in the production of cultured fish and shellfish such as sea bream, salmon, and shrimp and the like. Astaxanthin also has uses in providing pigment to poultry (broiler skin and egg yolks), avian plumage, and ornamental fish such as Koi.
Astaxanthin has also been found to have diverse biological functions. It is a vitamin A precursor, acts as a scavenger and/or quencher of free radicals and active oxygen species, and has been shown to enhance the immune response. See Misawa et al., J. Bacteriol., 177:6575-6584 (1995). From studies of the properties of astaxanthin, it is a carotenoid of great interest to the pharmaceutical, “nutraceutical” (as a pre-cursor to vitamin A and other properties), and food industries. The complete biomedical properties of astaxanthin remain to be elucidated, but initial results suggest that it could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system and as an anti-inflammatory agent. See Tanaka et al., Carcinogenesis, 15(1):15-19 (1994); Jyonouchi et al., Nutrition and Cancer, 19(3):269-280 (1993); and Jyonouchi et al., Nutrition and Cancer, 16(2):93-105 (1991).
U.S. Pat. No. 6,433,025 to Lorenz teaches that astaxanthin can be used to retard or prevent sunburn when administered at an amount of about 1 to about 100 milligrams (mg) per day, and preferably about 2 to about 10 mg per day. The astaxanthin can be administered in a composition by a peroral, topical or injectable route.
Sources of astaxanthin include crustaceans such as a krill in the Antarctic Ocean, cultured products of the yeast Phaffia rhodozyma, cultured products of a green alga Haematococcus pluvialis, and products obtained by organic synthetic methods. However, when crustaceans such as krill or the like are used, a great deal of work and expense are required for the isolation of astaxanthin from contaminants such as lipids and the like during the harvesting and extraction. Moreover, in the case of the cultured product of the yeast Phaffia, a great deal of expense is required for the gathering and extraction of astaxanthin because the yeast has rigid cell walls and produces astaxanthin only in a low yield.
H. pluvialis produces one of the highest levels of astaxanthin (0.5-2 percent dry weight) among organisms, with the astaxanthin synthesized by this alga being present mostly as the mono-long chain ester (70% of the carotenoid fraction), along with a smaller amount of di-long chain ester (10% of the carotenoid fraction). Free astaxanthin (5% of the carotenoid fraction) is also present as are β-carotene, canthaxanthin and lutein. See, BioAstin/NatuRose™ Technical Bulletin #015, Cyanotech Corporation (2001). Extraction from H. pluvialis is difficult because of cell wall properties of this alga. Furthermore, H. pluvialis needs high light levels for astaxanthin formation and has to be cultured under special conditions.
For these reasons, astaxanthin produced from biological sources is often deemed to be inferior to that obtained by the organic synthetic methods on the basis of cost. The organic synthetic methods however have a problem of by-products produced during the synthesis because of use of astaxanthin as a feed for fish and shellfish, and as an additive to foods. The products obtained by the organic synthetic methods can be contrary to some consumer's preference for naturally produced products.
Thus, there is a need for a natural and economic source of astaxanthin that consumers regard as desirable. It would be desirable to supply an inexpensive astaxanthin that is free from contaminating side products and is produced from a biological source.
Astaxanthin and other ketocarotenoids also occur in certain plants, especially certain species belonging to the family Ranunculaceae and genus Adonis. Adonis vernalis, Adonis aestivalis and Adonis palaestina are species of the genus Adonis that produce astaxanthin in their flower petals.
Species of the genus Adonis also produce mixtures of cardenolides in their flowers. Because the cardenolides are produced in the flowers, the flowers cannot be fed without pre-treatment to fish or other animals because the cardenolides are toxic to fish and to other animals at high concentrations. Therefore, there is a need to reduce or eliminate cardenolides from astaxanthin preparations derived from Adonis. 
Adonis is the common name for dried plant material, extracts and tinctures containing astaxanthin and cardenolides from the yellow-flowered perennial species Adonis vernalis. The cardenolides are used in herbal medicine (“phytomedicine”) and homoeopathy for the treatment of heart arrhythmia and kidney disease. Cardenolides act very much like digitalis (from foxglove; widely used in conventional as well as alternative medicine). Adonis preparations have the advantages of acting more rapidly than digitalis and are non-cumulative.
Germany and France are the principal consumers of Adonis in herbal medicine, whereas Bulgaria, Hungary, Russia, Romania and the Ukraine are the major suppliers. The vast majority of A. vernalis harvested is collected from the wild, rather than being cultivated, particularly in Russia, Romania and the Ukraine.
By 1998, unsustainable harvesting in Russia had reduced A. vernalis stocks to dangerously low levels. A vernalis is now included in the Red Data books (listing endangered species) of most of the countries comprising its range, and harvesting from the wild is prohibited by law in a number of countries.
Domesticating species of the genus Adonis or trying to achieve large-scale cultivation of A. vernalis is very difficult because of problems obtaining either self- or cross-pollinated seed, problems associated with poor germination (including almost complete loss of seed viability following storage for 12 months), delay in reaching harvestable flower size (up to 4 years from sowing), and low yields. Another problem with many Adonis species is that they have to be harvested by hand to insure the collection of flowers and not other parts of the plant. Plants currently cannot be grown in large numbers or the flowers harvested mechanically.
U.S. Pat. No. 5,453,565 to Mawson discloses a new strain of Adonis aestivalis that is said to have an average of 18-22 petals per flower head and contain an average of 200-350 μg of astaxanthin pigment per flower head. Methods of extracting astaxanthin are also disclosed. Seeds of that strain were said to be deposited on 18 Jul. 1990 with the National Collection of Industrial and Marine Bacteria Limited, Aberdeen under Accession No. NCIMB 40309, in accordance with the Budapest Treaty. The plants that mature from that deposited strain are referred to under the denomination Adonis aestivalis L. ‘Loders Red’. Those plants are also subject to Plant Breeders Rights that can be found at file number 19981166 of Aug. 28, 1998, grant number 6243 dated Jun. 5, 2000, that expires on Dec. 31, 2025.
Flowers of the species Adonis palaestina produce astaxanthin, other hydroxycarotenoids and hydroxyketocarotenoids primarily in the petals of the bright red flowers as di-long chain fatty acid esters. This species of the genus Adonis has heretofore not been rigorously examined as a commercial source of astaxanthin. The present inventors have now examined this species and found it to be readily improvable to provide for the commercial production of astaxanthin diesters and mixtures of those diesters with other carotenoids. The description that follows describes several of these improved plants.